C-Reactive Protein ("CRP" herein) is normally a trace constituent in blood where the serum concentration in healthy adults normally remains below 5 mg/l. After the onset of a stimulus, such as, tissue injury, inflammation, and various infections, the concentration rapidly and dramatically rises several thousand-fold or more. Upon healing or recovery of the patient and subsequent reduction of the stimulus, the concentration of CRP returns to the trace value. Monitoring disease activity by measuring the concentration level of CRP has become a much used practice in clinical chemistry.
One type of noncompetitive assay particularly useful for the measurement of CRP involves the formation of a "sandwich" of the ligand between two specific binding receptors (and thus is commonly referred to as a sandwich assay). One of the receptors is attached to a water insoluble substrate and the other receptor is commonly labeled with a substance that generates a measurable signal. The two receptors typically recognize two spatially distinct sites on the ligand. Because one of the receptors is attached to the insoluble support, the entire "sandwich" of ligand and two receptors is also insoluble once formed. Consequently, the insoluble "sandwich" may be easily separated from soluble materials (including the unreacted labeled receptor). After separation, either the insoluble "sandwich" may measured (or the unbound labeled receptor may be measured) to indicate the concentration of the ligand.
The receptors in sandwich assays may be various components capable of specifically binding with the targeted C-Reactive Protein. Typically, antibodies specific to CRP as well as modified phosphorycholine (as disclosed in UK Patent Application 2217335A) have been used as receptors in immunoassay systems specific to CRP.
One problem that has been observed in using sandwich assays for the detection of CRP is the potential misinterpretation of results generated by the signal system of the assay. It is particularly difficult to measure (and detect) CRP because of the ligand's widely varying concentration range from trace values to increases of several thousand fold. A "hook" effect has been associated with CRP assays when the CRP is present at high concentrations. As familiar to those skilled in the art, when the CRP is present at high concentrations, the assay may give a false negative result that the CRP is present in low amounts although the CRP concentration is actually very high. The term "hook" describes the calibration curve of the assay having the false negative, where the calibration line resembles a hook. For obvious reasons, reliance on an assay having a "hook" effect is potentially hazardous.
Cragle et al (U.S. Pat. No. 4,595,661 issued Jun. 17, 1986) describe an immunoassay method of reducing the "hook" effect by employing low affinity antibodies in addition to the two receptors typically used.
Although many advances have been made in specific binding assays, it is highly desirable to discover an alternative specific binding sandwich assay system that is capable of improving the measurement of CRP.